The present invention is in the field of compositions having RNA-cleavage activity.
Hammerhead ribozymes are an example of catalytic RNA molecules which are able to recognize and cleave a given specific RNA substrate (Hotchins et al., Nucleic Acids Res. 14:3627 (1986); Keese and Symons, in Viroids and viroid--like pathogens (J. J. Semanchik, publ., CRC-Press, Boca Raton, Fla., 1987), pages 1-47). The catalytic center of hammerhead ribozymes is flanked by three stems and can be formed by adjacent sequence regions of the RNA or also by regions which are separated from one another by many nucleotides. FIG. 1 shows a diagram of such a catalytically active hammerhead structure. The stems have been denoted I, II and III. The nucleotides are numbered according to the standard nomenclature for hammerhead ribozymes (Hertel et al., Nucleic Acids Res. 20:3252 (1992)). In this nomenclature, bases are denoted by a number which relates their position relative to the 5' side of the cleavage site. Furthermore, each base that is involved in a stem or loop region has an additional designation (which is denoted by a decimal point and then another number) that defines the position of that base within the stem or loop. A designation of N.sup.11.3 would indicate that this base is involved in a paired region and that it is the third base in that stem going away for the core region. This accepted convention for describing hammerhead derived ribozymes allows for the nucleotides involved in the core of the enzyme to always have the same number relative to all of the other nucleotides. The size of the stems involved in substrate binding or core formation can be any size and of any sequence, and the position of A.sup.9, for example, will remain the same relative to all of the other core nucleotides. Nucleotides designated, for example, N .sup.12 or N.sup.9 represent an inserted nucleotide where the position of the carrot ( ) relative to the number denotes whether the insertion is before or after the indicated nucleotide. Thus, N .sup.12 represents a nucleotide inserted before nucleotide position 12, and N.sup.9 represent a nucleotide inserted after nucleotide position 9.
The consensus sequence of the catalytic core structure is described by Ruffner and Uhlenbeck (Nucleic Acids Res. 18:6025-6029 (1990)). Perrirnan et al. (Gene 113:157-163 (1992)) have meanwhile shown that this structure can also contain variations, for example, naturally occurring nucleotide insertions such as N.sup.9 and N .sup.12. Thus, the positive strand of the satellite RNA of the tobacco ring-spot virus does not contain any of the two nucleotide insertions while the +RNA strand of the virusoid of the lucerne transient streak virus (vLTSV) contains a N.sup.9 =U insertion which can be mutated to C or G without loss of activity (Sheldon and Symons, Nucleic Acids Res. 17:5679-5685 (1989)). Furthermore, in this special case, N.sup.7 =A and R.sup.15.1 =A. On the other hand, the minus strand of the carnation stunt associated viroid (-CarSV) is quite unusual since it contains both nucleotide insertions, that is N .sup.12 =A and N.sup.9 =C (Hernandez et al., Nucleic Acids Res. 20:6323-6329 (1992)). In this viroid N.sup.7 =A and R.sup.15.1 =A. In addition, this special hammerhead structure exhibits a very effective self-catalytic cleavage despite the more open central stem.
Possible uses of hammerhead ribozymes include, for example, generation of RNA restriction enzymes and the specific inactivation of the expression of genes in, for example, animal, human or plant cells and prokaryotes, yeasts and plasmodia. A particular biomedical interest is based on the fact that many diseases, including many forms of tumors, are related to the overexpression of specific genes. Inactivating such genes by cleaving the associated mRNA represents a possible way to control and eventually treat such diseases. Moreover there is a great need to develop antiviral, antibacterial and antifungal pharmaceutical agents. Ribozymes have potential as such anti-infective agents since viral expression can be blocked selectively by cleaving viral or microbial RNA molecules vital to the survival of the organism can be selectively destroyed.
In addition to needing the correct hybridizing sequences for substrate binding, substrates for hammerhead ribozymes have been shown to strongly prefer the triplet N.sup.16.2 U.sup.16.1 H.sup.17 where N can be any nucleotide, U is uridine, and H is either adenosine, cytidine, or uridine (Koizumi et al., FEBS Lett. 228, 228-230 (1988); Ruffner et al., Biochemistry 29, 10695-10702 (1990); Perriman et al., Gene 113, 157-163 (1992)). The fact that changes to this general rule for substrate specificity result in non-functional substrates implies that there are "non core compatible" structures which are formed when substrates are provided which deviate from the stated requirements. Evidence along these lines was recently reported by Uhlenbeck and co-workers (Biochemistry 36:1108-1114 (1997)) when they demonstrated that the substitution of a G at position 17 caused a functionally catastrophic base pair between G.sup.17 and C.sup.3 to form, both preventing the correct orientation of the scissile bond for cleavage and the needed tertiary interactions of C.sup.3 (Murray et al., Biochem. J. 311:487-494 (1995)). The strong preference for a U at position 16.1 may exist for similar reasons. Many experiments have been done in an attempt to isolate ribozymes which are able to efficiently relieve the requirement of a U at position 16.1, however, attempts to find hammerhead type ribozymes which can cleave substrates having a base other than a U at position 16.1 have proven impossible (Perriman et al., Gene 113, 157-163 (1992)).
Efficient catalytic molecules with reduced or altered requirements in the cleavage region are highly desirable because their isolation would greatly increase the number of available target sequences that molecules of this type could cleave. For example, it would be desirable to have a ribozyme variant that could efficiently cleave substrates containing triplets other than N.sup.16.2 U.sup.16.1 H.sup.17 since this would increase the number of potential target cleavage sites.
Chemically modified oligonucleotides which contain a block of deoxyribonucleotides in the middle region of the molecule have potential as pharmaceutical agents for the specific inactivation of the expression of genes (Giles et al., Nucleic Acids Res. 20:763-770 (1992)). These oligonucleotides can form a hybrid DNA-RNA duplex in which the DNA bound RNA strand is degraded by RNase H. Such oligonucleotides are considered to promote cleavage of the RNA and so cannot be characterized as having an RNA-cleaving activity nor as cleaving an RNA molecule (the RNase H is cleaving). A significant disadvantage of these oligonucleotides for in vivo applications is their low specificity, since hybrid formation, and thus cleavage, can also take place at undesired positions on the RNA molecules.
Previous attempts to recombinantly express catalytically active RNA molecules in the cell by transfecting the cell with an appropriate gene have not proven to be very effective since a very high expression was necessary to inactivate specific RNA substrates. In addition the vector systems which are available now cannot generally be applied. Furthermore, unmodified ribozymes cannot be administered directly due to the sensitivity of RNA to degradation by RNases and their interactions with proteins. Thus, chemically modified active substances have to be used in order to administer hammerhead ribozymes exogenously (discussed, for example, by Heidenreich et al., J. Biol. Chem. 269:2131-2138 (1994); Kiehntopf et al., EMBO J. 13:4645-4652 (1994); Paolella et al., EMBO J. 11:1913-1919 (1992); and Usman et al., Nucleic Acids Symp. Ser. 31:163-164 (1994)).
U.S. Pat. No. 5,334,711 describes such chemically modified active substances based on synthetic catalytic oligonucleotide structures with a length of 35 to 40 nucleotides which are suitable for cleaving a nucleic acid target sequence and contain modified nucleotides that contain an optionally substituted alkyl, alkenyl or alkynyl group with 1-10 carbon atoms at the 2'-O atom of the ribose. These oligonucleotides contain modified nucleotide building blocks and form a structure resembling a hammerhead structure. These oligonucleotides are able to cleave specific RNA substrates. Examples of oligonucleotides are described having an active center which has a length of 14 nucleotides and which contains several ribonucleotides. These ribonucleotides increase the sensitivity of the oligonucleotide to enzymes which cleave RNA. A further disadvantage is the length of the active center which can often lead to unspecific hybridization.
WO 95/11304 describes RNA-cleaving nucleic acids with an active center that is free of ribonucleotide building blocks but instead contains deoxyribonucleotides. However, the deoxyribonucleotides used in the active center result in a very low RNA cleavage activity. Thus, it was reported that a 13-mer deoxyribozyme of the "GAAA" type based on LTSV was not able to cleave a 41-mer oligoribonucleotide substrate while the corresponding 13-mer ribozyme exhibited catalytic activity (Jeffries and Symons, Nucleic Acids Res. 17:1371-1377 (1989)).
WO 97/18312 describes oligomers which contain only part of a catalytic core resembling a hammerhead catalytic core. These oligomers, when associated with an RNA substrate having a motif resembling the complementary part of a catalytic core, induce cleavage of the RNA substrate. The RNA substrates for use with these oligomers all have a U at position 16.1.
The use of a large number of deoxyribonucleotides in the hybridization arms or in the active center can lead to a loss of specificity due to an activation of RNase H since sequences which are related to the desired target sequence can also be cleaved. Moreover, catalytic DNA oligomers are not particularly well suited for in vivo applications due to interactions with proteins, and lack of resistance to degradation by nucleases.
The shortest ribozymes that have been previously used have a minimum length of 15+N+M nucleotides, the active center being 15 nucleotides long and N and M being the length of the recognition sequences (Benseler et al., J. Am. Chem. Soc. 115:8483-8484 (1993)). Such ribozymes also contain ribonucleotides in at least five positions of the catalytic center (Paolella et al., EMBO J. 11:1913-1919 (1992), and Yang et al., Bio-chemistry 31:5005-5009 (1992)).
It is therefore an object of the present invention to provide compositions that induce cleavage of RNA, and in particular to provide oligomers that induce cleavage of RNA and which at the same time have a high stability, activity, and specificity.
It is another object of the present invention to provide compositions that induce cleavage of RNA substrates having a cleavage site triplet other than N.sup.16.2 U.sup.16.1 H.sup.17.